Surface modifying composition

ABSTRACT

Alkanethiols of formula (1) and the enantimomers of the alkanethiol of formula (1): 
 
HS-L-Q-T  (1), 
and disulfides of formula (3) and the enantimomers of the disulfide of formula (3): 
 
T-Q-L-S—S-J  (3), 
 
where -T is a moiety of formula (2)  
                 
 
R 1  and R 2  are each individually selected from the group consisting of H and OH; a is 0 to 3; b is 0 to 3; and   indicates that the chirality of the carbon atom to which it is attached is either R or S; may form inert surfaces that prevent the unwanted adsorption of proteins and cells.

This application is a continuation of U.S. patent application Ser. No.09/689,263, filed on Oct. 11, 2000.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The subject matter of this application was in part funded by the DARPAand the NIH (Grant no. OM-R29-54621-04). The government may have certainrights in this invention.

BACKGROUND

The present invention relates to self-assembled monolayers (SAMs) ofalkanethiolates.

SAMs of alkanethiolates on gold are an important class of modelsubstrates for mechanistic studies of the interactions of proteins andcells with surfaces. A primary reason for the importance of these modelsubstrates is the availability of SAMs that are inert—that is, SAMs thatprevent or inhibit the adsorption of protein and the attachment ofcells. Monolayers terminated in short oligomers of the ethylene glycolgroup ([OCH₂CH₂]_(n)OH, n=3-6) are known to prevent the adsorption ofvirtually all proteins under a wide range of conditions. This class ofSAMs has been critical for developing methods to pattern the adhesion ofcells, to prepare surfaces that present ligands for selectiveinteractions with proteins, and to design electroactive dynamicsubstrates that can modulate the selective recognition of proteins.

Given the importance of inert surfaces, it is surprising that there areso few functional groups that have been identified that render materialsinert—and none that are as effective at preventing protein adsorption asare oligo(ethylene glycol) groups when presented on SAMs. A monolayerpresenting a tri(propylene sulfoxide) group was reported as inert toprotein adsorption and cell attachment. While monolayers terminated inthis propylene sulfoxide group prevented protein adsorption, they weregenerally less effective and less stable over time than were SAMspresenting oligo(ethylene glycol) groups. The synthetic route to thepropylene sulfoxide oligomers is laborious, which further limits theutility of these monolayers. Another example, a monolayer terminated inthe carbohydrate maltose, was reported to prevent protein adsorption. Inthat work, the lack of adsorption was demonstrated with ellipsometricmeasurements, which required the substrate be removed from solution,rinsed and then dried prior to the measurement. Because this and relatedex situ techniques cannot detect weak and reversible adsorption, it isnot clear whether these monolayers will ultimately prove inert insettings where the substrate is in contact with a protein-containingsolution for long periods of time.

The development or identification of alternative functional groups thatcan render SAMs inert to protein adsorption and cell attachment isimportant, since it is probable that no one surface chemistry will bebest suited to give an inert interface under all conditions. Theavailability of several inert surface chemistries would permitoptimization of the substrate properties for each unique application.

BRIEF SUMMARY

In a first aspect, the present invention is an alkanethiol of formula(1) and the enantimomers of the alkanethiol of formula (1):HS-L-Q-T  (1),where -L- is -(A_(x)-B_(y)-E_(z)-D)_(w)-; each A, B, E and D areindividually C(R_(A)R_(A)′)—, —C(R_(B)R_(B)′)—, —C(R_(E)R_(E)′)—, and—C(R_(D)R_(D)′)—, respectively; each R_(A), R_(B), R_(E) and R_(D) areindividually H, or any two of R_(A), R_(B), R_(E) and R_(D) togetherform a bond, or R_(A), R_(B), R_(E) and R_(D) together with the atoms towhich they are bonded form a six-membered aromatic ring; each R_(A)′,R_(B)′, R_(E)′ and R_(D)′ are individually H, or any two of R_(A)′,R_(B)′, R_(E)′ and R_(D)′ together form a bond, or R_(A)′, R_(B)′,R_(E)′ and R_(D)′ together with the atoms to which they are bonded forma six-membered aromatic ring; each x, y and z are individually either 0or 1; w is 1 to 5; -Q- is selected from the group consisting of

-T is a moiety of formula (2)

R¹ and R² are each individually selected from the group consisting of Hand OH; a is 0 to 3; b is 0 to 3;

and indicates that the chirality of the carbon atom to which it isattached is either R or S.

In a second aspect, the present invention is a disulfide of formula (3)and the enantimomers of the disulfide of formula (3):T-Q-L-S—S-J  (3),where -L-, -Q-, and -T have the same meaning as above; -J is selectedfrom the group consisting of H, halogen, R, —OR, —NRR′, —C(O)R, and—C(O)OR; R is selected from the group consisting of alkyl, alkenyl,alkynyl, aryl and heterocyclic radical; and R′ is selected from thegroup consisting of H, alkyl, alkenyl, alkynyl, aryl and heterocyclicradical.

In a third aspect, the present invention is a substrate, includes (i) asurface layer comprising gold, and (ii) a plurality of moieties, on atleast a portion of the surface layer, where the moieties arealkanethiolate moieties of formula (5) and enantimomers of thealkanethiolate moieties of formula (5):Surf-S-L-Q-T  (5);-L-, -Q-, and -T have the same meaning as above; and Surf designateswhere the moiety attaches to the surface.

In a fourth aspect, the present invention is a substrate, including (i)a surface layer comprising gold, and (ii) a monolayer comprisingmoieties, on at least a portion of the surface layer, where the moietiesare alkanethiolate moieties; and the monolayer does not fail a cellpatterning test at 25 days.

In a fifth aspect, the present invention is a cell chip, including asubstrate described above, and cells on the substrate.

In a sixth aspect, the present invention is a protein chip, including asubstrate described above, and protein on the substrate.

Definitions

“Alkyl” (or alkyl- or alk-) refers to a substituted or unsubstituted,straight, branched or cyclic hydrocarbon chain, preferably containingfrom 1 to 20 carbon atoms. More preferred alkyl groups are alkyl groupscontaining from 7 to 16 carbon atoms. Preferred cycloalkyls have from 3to 10, preferably 3-6, carbon atoms in their ring structure. Suitableexamples of unsubstituted alkyl groups include methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec-butyl,cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like.“Alkylaryl” and “alkylheterocyclic” groups are alkyl groups covalentlybonded to an aryl or heterocyclic group, respectively.

“Alkenyl” refers to a substituted or unsubstituted, straight, branchedor cyclic, unsaturated hydrocarbon chain that contains at least onedouble bond, and preferably 2 to 20, more preferably 7 to 16, carbonatoms. Exemplary unsubstituted alkenyl groups include ethenyl (orvinyl)(—CH═CH₂), 1-propenyl, 2-propenyl (or allyl)(—CH₂—CH═CH₂),1,3-butadienyl (—CH═CHCH═CH₂), 1-butenyl (—CH═CHCH₂CH₃), hexenyl,pentenyl, 1,3,5-hexatrienyl, and the like. Preferred cycloalkenyl groupscontain five to eight carbon atoms and at least one double bond.Examples of cycloalkenyl groups include cyclohexadienyl, cyclohexenyl,cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl,cycloheptadienyl, cyclooctatrienyl and the like.

“Alkynyl” refers to a substituted or unsubstituted, straight, branchedor cyclic unsaturated hydrocarbon chain containing at least one triplebond, and preferably 2 to 20, more preferably 7 to 16, carbon atoms.

“Aryl” refers to any monovalent aromatic carbocyclic or heteroaromaticgroup, preferably of 3 to 10 carbon atoms. The aryl group can bemonocyclic (i.e. phenyl (or Ph)) or polycyclic (i.e. naphthyl) and canbe unsubstituted or substituted. Preferred aryl groups include phenyl,naphthyl, furyl, thienyl, pyridyl, indolyl, quinolinyl or isoquinolinyl.

“Halogen” (or halo-) refers to fluorine, chlorine, iodine or bromine.The preferred halogen is fluorine or chlorine.

“Heterocyclic radical” refers to a stable, saturated, partiallyunsaturated, or aromatic ring, preferably containing 5 to 10, morepreferably 5 or 6, atoms. The ring can be substituted 1 or more times(preferably 1, 2, 3, 4 or 5 times) with a substituent. The ring can bemono-, bi- or polycyclic. The heterocyclic group consists of carbonatoms and from 1 to 3 heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. The heteroatoms can beprotected or unprotected. Examples of useful heterocyclic groups includesubstituted or unsubstituted, protected or unprotected acridine,benzathiazoline, benzimidazole, benzofuran, benzothiophene,benzothiazole, benzothiophenyl, carbazole, cinnoline, furan, imidazole,1H-indazole, indole, isoindole, isoquinoline, isothiazole, morpholine,oxazole (i.e. 1,2,3-oxadiazole), phenazine, phenothiazine, phenoxazine,phthalazine, piperazine, pteridine, purine, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, quinazoline, quinoline,quinoxaline, thiazole, 1,3,4-thiadiazole, thiophene, 1,3,5-triazines,triazole (i.e. 1,2,3-triazole), and the like.

“Substituted” means that the moiety contains at least one, preferably1-3 substituent(s). Suitable substituents include hydrogen (H) andhydroxyl (—OH), amino (—NH₂), oxy (—O—), carbonyl (—CO—), thiol, alkyl,alkenyl, alkynyl, alkoxy, halo, nitrile, nitro, aryl and heterocyclicgroups. These substituents can optionally be further substituted with1-3 substituents. Examples of substituted substituents includecarboxamide, alkylmercapto, alkylsulphonyl, alkylamino, dialkylamino,carboxylate, alkoxycarbonyl, alkylaryl, aralkyl, alkylheterocyclic, andthe like.

“Disulfide” means a compound containing a bond between two sulfur atoms.

“Alkanethiol” means a compound containing an alkyl group bonded to an SHgroup.

“Alkanethiolate” means a moiety corresponding to an alkanethiol withoutthe hydrogen of the SH group.

“Alkylene” refers to a substituted or unsubstituted, straight, branchedor cyclic hydrocarbon chain, preferably containing from 1 to 20 carbonatoms. More preferred alkylene groups are lower alkylene groups, i.e.,alkylene groups containing from 1 to 6 carbon atoms. Preferredcycloalkylenes have from 3 to 10, preferably 3-6, carbon atoms in theirring structure. Suitable examples of unsubstituted alkylene groupsinclude methylene, —(CH₂)_(n)—, —CH₂—CH(CH₃)—, —(C₆H₁₀)— where thecarbon atoms form a six-membered ring, and the like.

All other acronyms and abbreviations have the corresponding meaning aspublished in journals relative to the art of chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood from the following detailed description when considered inconnection with the accompanying drawings in which like referencecharacters designate like or corresponding parts throughout the severalviews and wherein:

FIG. 1 illustrates data from surface plasmon resonance spectroscopy forthe adsorption of fibrinogen to each of four SAMs, showing the relativechange in angle of minimum reflectivity as sloutions are flowed over theSAMs. The curves are offset vertically for clarity.

FIG. 2 is optical micrographs for cells patterned into circular regionsof HDT surrounded by regions of other SAMs.

FIG. 3 illustrates a patterned substrate.

FIG. 4 illustrates another patterned substrate.

DETAILED DESCRIPTION

The present invention includes new alkanethiols and disulfides, and SAMsprepared from these compounds that are inert surfaces. The SAMs exhibitexcellent inertness to both protein adhesion and cell attachment,demonstrating their usefulness to prepare protein and cell chips.

The alkanethiols have the structure shown in formula (1) and theenantimomers of the structure shown in formula (1):HS-L-Q-T  (1)where -L- is -(A_(x)-B_(y)-E_(z)-D)_(w)-; each A, B, E and D areindividually C(R_(A)R_(A)′)—, —C(R_(B)R_(B)′)—, —C(R_(E)R_(E)′)—, and—C(R_(D)R_(D)′)—, respectively; each R_(A), RB, R_(E) and R_(D) areindividually H, or any two of R_(A), R_(B), R_(E) and RD together form abond, or R_(A), R_(B), R_(E) and R_(D) together with the atoms to whichthey are bonded form a six-membered aromatic ring; each R_(A)′, R_(B)′,R_(E)′ and R_(D)′ are individually H, or any two of R_(A)′, R_(B)′,R_(E)′ and R_(D)′ together form a bond, or R_(A)′, R_(B)′, R_(E)′ andR_(D)′ together with the atoms to which they are bonded form asix-membered aromatic ring; each x, y and z are individually either 0 or1; and w is 1 to 5;

-   -   -Q- is selected from the group consisting of    -   -T is a moiety of formula (2)        where R¹ and R² are each individually selected from the group        consisting of H and OH; a is 0 to 3; b is 0 to 3;        and indicates that the chirality of the carbon atom to which it        is attached may be either R or S.

The disulfides have the structure shown in formula (3) and theenantimomers of the structure shown in formula (3):T-Q-L-S—S-J  (3)where -L-, -Q- and -T have the same meaning as in formula (1), and -J isselected from the group consisting of H, halogen, R, —OR, —NRR′, —C(O)R,and —C(O)OR; R is selected from the group consisting of alkyl, alkenyl,alkynyl, aryl and heterocyclic radical; R′ is selected from the groupconsisting of H, alkyl, alkenyl, alkynyl, aryl and heterocyclic radical.

Preferably, -L- contains 6 to 20 carbon atoms, more preferably 8 to 18carbon atoms. Preferably, -L- contains 1 or 0 double bonds, or 1 triplebond. Most preferably, -L- is an alkylene containing 6 to 18 carbonatoms.

Preferably, -Q- is —O— or —CH₂—.

Preferably, -T is a moiety of formula (2′)

Most preferably a is 1, b is 1 and at least one of R¹ and R² is OH.

Preferably, -J is a moiety of formula (4)L′-Q′-T′  (4),an alkyl having 1 to 4 carbon atoms, or is —(CH₂)_(c)(OCH₂CH₂)_(n)OH;where -L′-, -Q′-, and -T′ have the same meaning as -L-, -Q-, and -Trespectively, c is 2 to 20, and n is 1 to 3. Most preferably -J ismoiety of formula (4′):-L-Q-T  (4′).

The alkanethiols and disulfides of the present invention may besynthesized using reagents and reaction well known to those of ordinaryskill in the art, such as those described in “Advanced OrganicChemistry” J. March (Wiley & Sons, 1994); and “Organic Chemistry” 4thed., Morrison and Boyd (Allyn and Bacon, Inc., 1983). For example, thefollowing reaction scheme may be used:

Further photolysis with thioacetic acid and AIBN(2,2′-azobisisobutyronitrile) in THF (tetrahydrofuran) forms thethioester of the alkanethiol of formula (1). Hydrolysis then givesalkanethiols of formula (1) with -Q- being —O—. Optionally, —OH groupsin -T may be protected using acetone, and deprotection may take placebefore, after, or during hydrolysis of the thioester. For alkanethiolsof formula (1) where -Q- is —NH—, Br in the above reaction scheme abovemay be replaced with NH₂, and the OH may be converted to a tosylate ormesylate. For alkanethiols of formula (1) where -Q- is —CH₂—, the OH maybe converted to a tosylate or mesylate, and Br converted to thecorresponding Grignard. For alkanethiols of formula (1) where -Q- is—CO—O—, Br in the above reaction scheme may be replaced with CO₂H. Foralkanethiols of formula (1) where -Q- is —O—CO—, Br in the abovereaction scheme may be replaced with OH, and the OH may be converted tothe corresponding acid. For alkanethiols of formula (1) where -Q- is—NH—CO—, Br in the above reaction scheme may be replaced with NH₂, andthe OH may be converted to the corresponding acid. For alkanethiols offormula (1) where -Q- is —CO—NH—, Br in the above reaction scheme may bereplaced with CO₂H, and the OH may be converted to the correspondingprimary amine (for example, by tosylation or mesylation followed byreaction with ammonia). For alkanethiols of formula (1) where -Q- is—NH—CO—NH—, Br in the above reaction scheme may be replaced with NH₂,and the OH may be converted to the corresponding isocyanate. Foralkanethiols of formula (1) where -Q- is —NH—CO—O—, Br in the abovereaction scheme may be replaced with —N═C═O to give an isocyanate, whichis then reacted with the hydroxyl as shown. Similarly, the disulfidesmay be formed by first forming alkanethiols, followed by oxidativecoupling. When the disulfide is not symmetric, two differentalkanethiols are oxidized together.

When applied to a surface containing gold, the alkanethiols anddisulfides will form SAMs. In the case of the alkanethiols, the hydrogenis lost, and the remaining moiety attaches to the surface through thesulfur atom. In the case of the disulfides, the disulfide bridge isbroken, and the remaining moieties attach to the surface through thesulfur atoms. The surface preferably has a plurality of alkanethiolatemoieties shown in formula (5)Surf-S-L-Q-T  (5)where -L-, -Q- and -T have the same meaning as in formula (1), and Surfdesignates where the moiety attaches to the surface. The density ofmoieties on the surface is typically 10¹⁰±5% per square centimeter. Themoieties of the present invention may cover the entire surface, or maybe patterned on the surface. Patterning may be carried out by, forexample, by microprinting, as described in Mrksich, M.; Dike, L. E.;Tien, J.; Ingber, D. E.; Whitesides, G. M., Experimental Cell Research1997 235, 305-313; Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G.M.; Ingber, D. E., Science 1997, 276, 1425-1428; and Mrksich, M.;Whitesides, G. M., TIBTECH. 1995, 13, 228-235.

Preferably the surface contains gold, more preferably the surfacecontains 50 to 100 atom percent gold. Preferably, the surface is pure orfine gold, or an alloy of gold with copper, silver, or a combinationthereof.

The surface may be on a base. The base may have the same composition asthe surface (for example a gold surface on a gold plate), or the surfacemay be, for example, a film, foil, sheet, or plate, on a base having adifferent composition. The base may be any material, such as metal,ceramic, plastic, or a natural material such as wood. Examples of basesinclude glass, quartz, silicon, transparent plastic, aluminum, carbon,polyethylene and polypropylene.

The surface material may be attached to the base by any of a variety ofmethods. For example, a film of the surface material may be applied tothe base by sputtering or evaporation. If the surface material is a foilor sheet, in could be attached with an adhesive. Furthermore, thesurface need not completely cover the base, but may cover only a portionof the base, or may form a pattern on the base. For example, portions ofthe base could be patterned by sputtering the base, covering thoseportions of the base where no surface material is desired. Thesepatterns may include an array of regions containing, or missing, thesurface material.

A cell chip is an array of regions containing cells on a surface,separated by regions containing no cells or cells at a much lowerdensity. Since the SAMs of the present invention are inert, it isdifficult for cells to attach. A cell chip may be prepared by applyingSAMs of the present invention on regions of the surface that are toremain free of cells (or is intended to have cells at a lower density).The remaining regions could be left uncovered, or could be cover withSAMs that are not inert. For example, FIG. 3 illustrates one possiblepattern, where circles 10 contain a SAM of hexadecanethiolate, and theremainder 20 of the surface is covered with a SAM of the presentinvention, all on a surface 2. Another example, FIG. 4 illustratesanother possible pattern, where squares 30 contain a SAM ofhexadecanethiolate, and regions 40 surrounding the squares contain a SAMof the present invention, all on a surface 2.

Once the surface is patterned as desired, the cells may be allowed toattach and proliferate in the regions not containing SAMs of the presentinvention, by contacting those regions with cells, and providing thenutrients and conditions necessary for the cells to proliferate.

One measure of the inertness of a surface is the number of day needed tofail a cell pattern test. A cell pattern test is carried out as follows:A surface is patterned into hydrophobic regions of hexadecanethiolate(HDT) and surrounded by the inert monolayer to be tested. The pattern isformed by microprinting. Albino 3T3-Swiss fibroblasts (American TypeCulture Collection) are harvested from culture dishes with trypsin-EDTA,washed, and resuspended in DMEM (Dulbecco's Modification of Eagle'sMedium) supplemented with 10% fetal bovine serum. The suspended cellsare added to wells (10,000/well) containing patterned surfaces immersedin DMEM supplemented with serum. Serum-supplemented medium is exchangedwith fresh medium after 12 hours and then every 5 days. Cell culturesare maintained at 37° C. and photographed daily. When the density of thecells in the test region is equal to or great than the density in theregions of hexadecanethiolate, the test is failed. Preferably, SAMs ofthe present invention do not fail the cell patterning test at 12 days,more preferably at 14 days, even more preferably at 25 days.

A protein chip is an array of regions containing protein, separated byregions containing no protein or protein at a much lower density. Sincethe SAMs of the present invention are inert, it is difficult forproteins to attach. In the same manner as a cell chip may be prepared, aprotein chip may be prepared by applying SAMs of the present inventionon regions of the surface that are to remain free of protein (orintended to have protein at a lower density). The remaining regionscould be left uncovered, or could be cover with SAMs that are not inert.The same variety of patterns is possible with protein chips as describedabove for cell chips; FIGS. 3 and 4 are two possible patterns. Theprotein chip may then be prepared by contacting the surface with thedesired protein or proteins.

Alternatively, a protein chip may be formed by covering both regions ofthe surface that are intended to have protein, and those that are notintended to have protein, with SAMs of the present invention. Thoseregions that are intended to have protein will also include analkanethiolate corresponding to an alkanethiol terminated in a specificmoiety. The protein to be attached is chosen to include a region thatwill capture the specific moiety, anchoring the protein to the chosenregions. For example, the specific moiety may be the conjugate ofglutathione and benzoquinone of formula (6)

and the protein may be a fusion protein of a protein of interest (suchas the peptide hemagglutinin (HA) and glutathione S-transferase (GST)(the fusion protein being designated as GST-HA). When this fusionprotein encounters the alkanethiolate terminating in the conjugate, theprotein is cross-linked to the alkanethiolate, anchoring the protein inplace. Since the SAM mostly contains the inert moiety of the presentinvention, other undesired proteins do not attach to the surface.

GST is commonly used as an affinity handle for the purification ofrecombinant proteins; the protein is expressed and purified as the GSTfusion protein, and then treated with a protease to remove the GSTdomain (see, for example, Smith, D. B.; Johnson, K. S. Gene 1988, 67,31). These fusion proteins could be used without synthetic modificationto form protein chips. An additional benefit of using GST fusionproteins together with SAMs formed from alkanethiols terminated with theconjugate of formula (6) is that the reactivity of the surface towardsimmobilization could be controlled electrochemically, by way ofreversible reduction of the quinone of formula (6) to the unreactivehydroquinone.

EXAMPLES

Two experimental procedures were used to evaluate the monolayers asinert surfaces. Surface plasmon resonance (SPR) spectroscopy measuresthe adsorption of protein to the monolayer. All SPR experiments wereperformed on a BIAcore 1000 instrument. Substrates were glued intoplastic BIACore cassettes with a two-part epoxy (DEVCON).Phosphate-buffered saline (PBS; 10 mM phosphate, 150 mM sodium chloride,pH 7.6) was degassed under vacuum and all protein solutions werefiltered through 0.45-μm filters before use. SPR is well suited forcharacterization of protein adsorption because it measures adsorption insitu and can detect weak, readily reversible protein adsorption. For allmonolayers, a panel of five proteins that spanned a range in molecularweight and pI were investigated.

In a second set of experiments, the ability of the monolayers to patternthe long-term adhesion of albino Swiss 3T3 fibroblast cells wasinvestigated. These experiments used monolayers that are patterned intohydrophobic regions of hexadecanethiolate (HDT) and surrounded by theinert monolayer. Cells initially attach only to the hydrophobic regionsand proliferate to completely fill those regions after 2-3 days inculture. The periods of time that the cells remain confined to thepattern were compared—because the surrounding monolayer preventsattachment—to assess the effectiveness of the inert monolayer. The cellpatterning experiments impose a more demanding environment than do theprotein adsorption experiments, and hence, provide a more stringent testof inertness.

Protein adsorption and cell attachment on four different monolayers wascompared. Gold films were prepared by evaporation of titanium (1.5 nm)and then gold (40 nm) onto glass cover slips (0.20 mm, No. 2, Corning).The SAMs were prepared by immersing gold films of approximately 1 cm² inethanolic solutions of alkanethiol (2 mM) for 9 hours. The SAMs wererinsed with ethanol and dried with nitrogen before use. As a controlmonolayer that is not inert, a SAM of hexadecanethiolate that presentshydrophobic methyl groups at the surface were used. Monolayersterminated in the tri(ethylene glycol) group were used as the currentstandard for inert surfaces. Each of the two additional monolayerspresents the mannitol group and differs only in the length of alkylchain to which the mannitol group is appended.

Alkanethiol 1a has an alkyl chain of eleven methylene units, which isthe standard length in SAMs that are used in bio-interfacial science.Alkanethiol 1b has three fewer methylene units in the alkyl chain.Alkanethiol 2 terminates in the tri(ethylene glycol) group.

The synthetic scheme for preparing 1a and 1b is shown below. Thefollowing conditions were employed in the synthesis: (a) NaH/DMF; (b)CH₃COSH, AlBN, hv, THF; (c) HCl/CH₃OH, reflux. The reactions all useddistilled solvents and were performed under a nitrogen atmosphere.

Dimethylketal 3. To a solution of 1,2:3,4-di-o-isopropylidene-D-mannitol(316 mg, 1.20 mmol) in DMF (10 ml) was added sodium hydride (30 mg, 60%dispersion in oil, 1.20 mmol). The solution was stirred for 10 min atroom temperature and then 8-bromo-1-octene (220 mg, 1.20 mmol) was addedover a period of 5 min. The solution was stirred for 12 hours and thenconcentrated in vacuo. The residue was purified by flash chromatography(1.5% CH₃OH/CH₂Cl₂) to give 330 mg (0.883 mmol, 73%) of 3 as a colorlessoil: ¹H NMR (400 MHz, CDCl₃) δ 5.82-5.72 (m, 1H), 4.92 (q, J=10.2, 2H),4.41-4.30 (m, 2H), 4.12-3.95 (m, 3H), 3.77-3.65 (m, 2H), 3.13 (d,J=6.16, 1H), 2.00 (q, J=7.1, 2H), 1.53 (s, 3H), 1.42 (s, 3H), 1.40 (s,3H), 1.37 (s, 3H), 1.18 (m, 6H).

Thioester 4. A solution of 2 (151 mg, 0.41 mmol), thiol acetic acid (91mg, 1.2 mmol) and AIBN (80 mg, 0.487 mmol) in THF (15 ml) was irradiatedwith UV light (RAYONET PHOTOCHEMICAL REACTOR LAMP) in a photochemicalreactor for 5 hours with stirring. The solution was concentrated invacuo and the residue was purified by flash chromatography (1%CH₃OH/CH₂Cl₂) to give 147 mg (0.328 mmol, 82%) of 4 as a colorless oil:¹H NMR (400 MHz, CDCl₃) δ 4.41-4.30 (m, 2H), 4.12-3.95 (m, 3H),3.77-3.65 (m, 2H), 3.57-3.47 (m, 3H), 3.14-3.12 (d, J=6.16, 1H),2.90-2.85 (t, J=7.38, 2H), 2.33 (s, 3H), 1.64-1.55 (m, 4H), 1.53 (s,3H), 1.42 (s, 3H), 1.40 (s, 3H), 1.37 (s, 3H), 1.41 (m, 6H).

Alkanethiol 1b. To a solution of 4 (147 mg, 0.328 mmol) in methanol (15ml) was added 5 drops of 12 N HCl. The solution was refluxed for 8hours, cooled, and concentrated in vacuo. The residue was purified byrecrystallization from methanol to give 61 mg (0.187 mmol, 57%) of 1b asa white solid: ¹H NMR (400 MHz, CD₃OD) δ 3.85-3.59 (m, 6H), 3.58-3.46(m, 4H), 2.52-2.45 (t, 2H), 1.64-1.55 (m, 4H), 1.45-1.28 (m, 8H).

Alkanethiol 1a. The mannitol-terminated alkanethiol 1a was synthesizedwith the same protocol starting with 1′-bromo-1-undecene in place of8-bromo-1-octene.

SPR was used to measure the adsorption of five proteins—fibrinogen,pepsin, lysozyme, insulin, and trypsin—to the four monolayers. SPRmeasures changes in the refractive index of a solution near theinterface with a gold film by measuring changes in the angle (ΔΘ_(m)) atwhich p-polarized light reflected from the glass/gold interface has aminimum in intensity. SAMs were mounted in a flow cell and experimentswere performed by first flowing a phosphate buffered saline (PBS)through the cell for 5 minutes, then a solution of protein in the samebuffer (0.5 mg/ml) for 5 minutes, and then PBS for 5 minutes. The risein ΔΘ_(m) upon introduction of the protein solution has twocontributions. The first is due to the increase in refractive index ofthe solution caused by the dissolved protein—the “bulk effect”—and doesnot represent protein adsorption. The second contribution is due to theadsorption of protein to the SAM. The amount of protein that adsorbsirreversibly to the SAM is determined by comparing the shift in Θ_(m)before and after the SAM is exposed to protein. An increase in Θ_(m) of0.1° corresponds to an increase in density of adsorbed protein of 1ng/mm².

FIG. 1 shows data for the adsorption of fibrinogen to the four SAMs. Acomplete monolayer of protein adsorbs to the SAM of hexadecanethiolate.The change in Θ_(m) of 0.35° corresponds to a density of protein of3,500 pg/mm². The two monolayers presenting mannitol groups, bycontrast, show essentially no adsorption of fibrinogen. This lack ofadsorption is indistinguishable from that on monolayers presentingtri(ethylene glycol) groups. Experiments with the four other proteinsgave similar results and demonstrated that the mannitol group is broadlyeffective at preventing protein adsorption, as shown in Table 1. For allfive proteins, the amount of irreversible adsorption was less than 2% ofthe total amount that adsorbed on the methyl-terminated SAMs (HDT).TABLE 1 Adsorption of Protein on Monolayers. Fibrinogen^(b) PepsinLysozyme Insulin Trypsin Thick- 340 kD,^(d) 35 kD, 14 kD, 5.4 kD, 24 kD,SAM ness^(a) 5.5^(e) <1 1.4 5.4 10.5 1a 18.0 Å 27^(c) <10 19 15 <10 1b15.7 Å 45 12 10 25 16 2 19.5 Å 29 <10 <10 <10 <10 HDT 20.0 Å 3,432 1,3371,023 688 661^(a)Thickness of the SAM measured by ellipsometry (±0.5 Å).^(b)All protein solutions were 0.5 mg/ml in PBS (10 mM phosphate, 150 mMsodium chloride, pH = 7.6).^(c)The amount of protein that remains adsorbed to the SAM wasdetermined by SPR and is reported in the units of pg/mm². Each value ofabsorbed protein was taken from a single experiement and have a varienceof approximately 5% across independent experiments.^(d)Molecular weight of the protein.^(e)p/ of the protein.

The degree to which these monolayers could prevent the adhesion andgrowth of cells that were confined to a patterned substrate wasevaluated. Gold films were patterned by microcontact printing a set ofcircular regions of hexadecanethiolate that were 200 μm in diameter andthen the films were immersed in a solution of 1a, 1b or 2 to assemble aninert monolayer on the non-printed areas. Albino 3T3-Swiss fibroblasts(American Type Culture Collection) were harvested from culture disheswith trypsin-EDTA, washed, and resuspended in DMEM supplemented with 10%fetal bovine serum. The suspended cells were added to wells(10,000/well) containing patterned substrates immersed in DMEMsupplemented with serum. Serum-supplemented medium was exchanged withfresh medium after 12 hours and then every 5 days. Cell cultures weremaintained at 37° C. and photographed daily. When the suspension ofcells was added to culture wells containing the substrates, cellsattached only to the methyl-terminated regions. For each of thesubstrates patterned with the inert monolayers (1a, 1b and 2), cellsspread and proliferated to completely occupy these regions but did notinvade the surrounding inert regions. The period of time that cellsremain confined to the patterned monolayers provides a relative measureof the effectiveness of inert surface chemistries, and a direct measurefor identifying inert surfaces that are most effective for applicationsrequiring long-term cell patterning.

Albino 3T3-Swiss fibroblast cells patterned on the monolayers were keptat 37° C. in serum-containing media for four weeks. The media wasexchanged every five days and cells were photographed daily, as shown inFIG. 2. In all cases, cells remained patterned to the circular regionsof hexadecanethiolate for at least six days in culture. The tri(ethyleneglycol)-terminated (prepared from alkanethiol 2) monolayers began tofail after seven days, with cells spreading onto the inert regions.After 12 days, cells had migrated from the circular regions and dividedto give a confluent monolayer of cells, with a complete loss of pattern.Monolayers terminated in the mannitol group (prepared from alkanethiol1a) were substantially more effective at confining cells for longerperiods. Even after 21 days, cells remained completely confined to thepatterns. After 25 days in culture, the viability of the adherent cellsdecreased, and therefore prevented an assessment of the monolayer overlonger periods of time. Monolayers that presented the mannitol group onthe shorter polymethylene chain (prepared from alkanethiol 1b) were alsomore effective than monolayers presenting the tri(ethylene glycol)group, but were not as effective as monolayers of 1a. Monolayers of 1bgenerally failed at two weeks in culture. These long-term patterningexperiments were repeated on three separate occasions with consistentresults: monolayers of 2 fail at approximately one week, monolayers of1b fail at approximately two weeks and monolayers of 1a are effectivefor at least three weeks.

The SAMs 1a and 1b are highly effective as inert surfaces. SPR showedthat monolayers of 1a and 1b prevented the adsorption of severaldifferent proteins, including the “sticky” protein fibrinogen. In thisrespect, the monolayers were indistinguishable from monolayerspresenting tri(ethylene glycol) groups, which are the current standardfor inert model surfaces. When evaluated for the ability to maintain thepatterned adhesion of cells, it was found that SAMs of 1a were superiorto SAMs presenting tri(ethylene glycol) groups. In this and other work,it has been found that SAMs presenting tri(ethylene glycol) groups failat approximately seven days in culture. The SAMs of the presentinvention, by contrast, maintained the pattern of adherent cells over asmany as 25 days.

A reaction scheme to prepare an alkanethiol terminated in a moiety thatwill cross-link to a glutathione S-transferase fusion protein(alkanethiol (Z)) is described below. In the reaction scheme,hydroquinone was protected as the bis-tetrahydropyranyl ether, which wasthen deprotonated with tert-butyl lithium and alkylated with1,4-dibromobutane. Separately, molecule (Y) was prepared by protectingthe tri(ethylene glycol)-terminated alkanethiol as the tritylthioether.The free hydroxyl group of this molecule was alkylated with (X) to givethe conjugate. Deprotection of the tetrahydropyranyl ethers withpyridinium para-toluene sulfate revealed the hydroquinone, which wasthen oxidized with ammonium cerium nitrate to give the correspondingbenzoquinone. Michael addition of the cysteine thiol of glutathione tothe quinone resulted in the final precursor, which was treated withtrifluoroacetic acid to remove the trityl protecting group.

A monolayer was prepared from a mixture of alkanethiol (Z) and aalkanethiol 1a (in a ratio of 1:99). The surface was treated to oxidizethe hydroquinone group of alkanethiol (Z) to the correspondingbenzoquinone, either by applied electrical potentials (+500 mV for 1minute), or by treatment with a chemical oxidant (for example, with asubstituted quinone). Surface plasmon resonance (SPR) spectroscopy wasused to characterize the immobilization of GST-HA fusion protein to thismonolayer. We used GST-HA as a model system because an antibody againstthis peptide is available. In these experiments, phosphate bufferedsaline (pH 7.4) was flowed over the monolayer for 2 minutes to establisha baseline. A solution of GST-HA (100 □M) in the same buffer was thenflowed over the monolayer for 15 minutes to observe binding. Finally theprotein solution was replaced with buffer for 5 minutes to quantitatethe amount of protein that was irreversibly immobilized. The GST-HA wasefficiently immobilized to the monolayer and the anti-HA antibody boundto the immobilized peptide. This antibody did not bind, however, tomonolayers to which only GST had been immobilized, demonstrating thebiospecificity that is afforded with the inert SAMs of the presentinvention.

The SAMs of the present invention are significant because they extendthe time course over which cells can be patterned in culture. Thisenhancement is important for several applications that use cells asfunctional components. A primary example is the use of geneticallyengineered cells for screening libraries of drug candidates. This andother applications require substrates that can maintain patterned cellpopulations with excellent fidelity and over long periods of time.Furthermore, the SAMs of the present invention are highly effective atpreventing protein adsorption and extend the times over which culturedcells can be maintained in patterns.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. An alkanethiol of formula (1) and the enantimomers of the alkanethiolof formula (1):HS-L-Q-T  (1), where -L- is -(A_(x)-B_(y)-E_(z)-D)_(w)-; each A, B, Eand D are individually C(R_(A)R_(A)′)—, —C(R_(B)R_(B)′)—,—C(R_(E)R_(E)′)—, and —C(R_(D)R_(D)′)—, respectively; each R_(A), R_(B),R_(E) and R_(D) are individually H, or any two of R_(A), R_(B), R_(E)and R_(D) together form a bond, or R_(A), R_(B), R_(E) and R_(D)together with the atoms to which they are bonded form a six-memberedaromatic ring; each R_(A)′, R_(B)′, R_(E)′ and R_(D)′ are individuallyH, or any two of R_(A)′, R_(B)′, R_(E)′ and R_(D)′ together form a bond,or R_(A)′, R_(B)′, R_(E)′ and R_(D)′ together with the atoms to whichthey are bonded form a six-membered aromatic ring; each x, y and z areindividually either 0 or 1; w is 1 to 5; -Q- is selected from the groupconsisting of

-T is a moiety of formula (2)

R¹ and R² are each individually selected from the group consisting of Hand OH; a is 0 to 3; b is 0 to 3; and

indicates that the chirality of the carbon atom to which it is attachedis either R or S.
 2. The alkanethiol of claim 1, where -T is a moiety offormula (2′)


3. The alkanethiol of claim 2, where -Q- is —O— or —CH₂— and
 4. Adisulfide of formula (3) and the enantimomers of the disulfide offormula (3):T-Q-L-S—S-J  (3), where -L- is -(A_(x)-B_(y)-E_(z)-D)_(w)-; each A, B, Eand D are individually C(R_(A)R_(A)′)—, —C(R_(B)R_(B)′)—,—C(R_(E)R_(E)′)—, and —C(R_(D)R_(D)′)—, respectively; each R_(A), R_(B),R_(E) and R_(D) are individually H, or any two of R_(A), R_(B), R_(E)and R_(D) together form a bond, or R_(A), R_(B), R_(E) and R_(D)together with the atoms to which they are bonded form a six-memberedaromatic ring; each R_(A)′, R_(B)′, R_(E)′ and R_(D)′ are individuallyH, or any two of R_(A)′, R_(B)′, R_(E)′ and R_(D)′ together form a bond,or R_(A)′, R_(B)′, R_(E)′ and R_(D)′ together with the atoms to whichthey are bonded form a six-membered aromatic ring; each x, y and z areindividually either 0 or 1; w is 1 to 5; -Q- is selected from the groupconsisting of

-T is a moiety of formula (2)

R¹ and R² are each individually selected from the group consisting of Hand OH; a is 0 to 3; b is 0 to 3;

indicates that the chirality of the carbon atom to which it is attachedis either R or S; -J is selected from the group consisting of H,halogen, R, —OR, —NRR′, —C(O)R, and —C(O)OR; R is selected from thegroup consisting of alkyl, alkenyl, alkynyl, aryl and heterocyclicradical; and R′ is selected from the group consisting of H, alkyl,alkenyl, alkynyl, aryl and heterocyclic radical.
 5. The disulfide ofclaim 4, where -J is a moiety of formula (4):-L′-Q′-T′  (4), an alkyl having 1 to 4 carbon atoms, or—(CH₂)_(c)(OCH₂CH₂)_(n)OH; and where -L′- is-(A_(x)-B_(y)-E_(z)-D)_(w)-; -Q′- is selected from the group consistingof

-T′ is a moiety of formula (2)

c is 2 to 20, and n is 1 to
 3. 6. The disulfide of claim 5, where -J isa moiety of formula (4′):-L-Q-T  (4′).
 7. The disulfide of claim 6, where -T is a moiety offormula (2′)


8. The disulfide of claim 7, where -Q- is —O— or —CH₂—.
 9. A substrate,comprising: (i) a surface layer comprising gold, and (ii) a plurality ofmoieties, on at least a portion of the surface layer, where the moietiesare alkanethiolate moieties of formula (5) and enantimomers of thealkanethiolate moieties of formula (5):Surf-S-L-Q-T  (5); -L- is -(A_(x)-B_(y)-E_(z)-D)_(w)-; each A, B, E andD are individually C(R_(A)R_(A)′)—, —C(R_(B)R_(B)′)—, —C(R_(E)R_(E)′)—,and —C(R_(D)R_(D)′)—, respectively; each R_(A), R_(B), R_(E) and R_(D)are individually H, or any two of R_(A), R_(B), R_(E) and R_(D) togetherform a bond, or R_(A), R_(B), R_(E) and R_(D) together with the atoms towhich they are bonded form a six-membered aromatic ring; each R_(A)′,R_(B)′, R_(E)′ and R_(D)′ are individually H, or any two of R_(A)′,R_(B)′, R_(E)′ and R_(D)′ together form a bond, or R_(A)′, R_(B)′,R_(E)′ and R_(D)′ together with the atoms to which they are bonded forma six-membered aromatic ring; each x, y and z are individually either 0or 1; w is 1 to 5; -Q- is selected from the group consisting of

-T is a moiety of formula (2)

R¹ and R² are each individually selected from the group consisting of Hand OH; a is 0 to 3; b is 0 to 3;

indicates that the chirality of the carbon atom to which it is attachedis either R or S; and Surf designates where the moiety attaches to thesurface.
 10. The substrate of claim 9, further comprising: (iii) amonolayer comprising the moieties, where the monolayer does not fail acell patterning test at 12 days.
 11. The substrate of claim 9, furthercomprising: (iv) a base, where the surface layer is on the base.
 12. Thesubstrate of claim 11, where -T is a moiety of formula (2′)


13. The substrate of claim 12, where -Q- is —O— or —CH₂—.
 14. Thesubstrate of claim 9, where the monolayer does not fail a cellpatterning test at 12 days.
 15. A cell chip, comprising: (A) thesubstrate of claim 9, and (B) cells, on the substrate.
 16. A method ofmaking the alkanethiol of claim 1, comprising: hydrolyzing a thioester,to form the alkanethiol of formula (1).
 17. A method of making thedisulfide of claim 4, comprising: oxidizing a first alkanethiol, to formthe disulfide of formula (3).
 18. A method of making a substrate,comprising contacting a surface with the alkanethiol of claim 1; wherethe surface comprises gold.
 19. A method of making a substrate,comprising contacting a surface with the disulfide of claim 4; where thesurface comprises gold.
 20. A method of making a cell chip, comprising:contacting cells with the substrate of claim 9.